Centre for Drug Research Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Finland Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation Teemu Nissilä ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki, for public examination in Auditorium 1041, Viikki Biocentrum 2, on 27 May 2011, at 12 noon. Helsinki 2011 Supervisors: Adjunct Professor Raimo A. Ketola Centre for Drug Research Faculty of Pharmacy University of Helsinki Finland Professor Risto Kostiainen Division of Pharmaceutical Chemistry Faculty of Pharmacy University of Helsinki Finland Reviewers: Professor Hubert Girault Laboratory of Physical and Analytical Electrochemistry École polytechnique fédérale de Lausanne Switzerland Adjunct Professor Ilkka Ojanperä Hjelt Institute Department of Forensic Medicine University of Helsinki Finland Opponent: Professor Janne Jänis Department of Chemistry Faculty of Science and Forestry University of Eastern Finland Finland © Teemu Nissilä 2011 ISBN 978-952-10-6963-5 (paperback) ISSN 1795-7079 ISBN 978-952-10-6964-2 (PDF) http://ethesis.helsinki.fi Helsinki University Print Helsinki 2011 II Abstract This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of micro- channels, which are divided to the sampling spot and the channel, which leads to a sharp electrospray tip. Microchannels contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on- chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry. III Acknowledgements This study was carried out in the Centre for Drug Research of the University of Helsinki, Finland. Most of the work was done in facilities of the Division of Pharmaceutical Chemistry during the years 2006 - 2011. I acknowledge The Academy of Finland (projects no. 111991 and 129633), TEKES (project MISIMA 440006) and Centre for Drug Research for financial support of this work and the Division of Pharmaceutical Chemistry for the study facilities. Thanks are also due for all the additional support that I received from Helsinki University Research funds and the graduate school of Chemical Sensors and Microanalytical Systems (CHEMSEM). There are many people I wish to acknowledge for their contributions to this work: First of all I want to thank Adjunct Professor Raimo Ketola and Professor Risto Kostiainen for the opportunity to work under their inspirational supervision and also for introducing me to the world of science; Especially the enthusiasm of Ketola, his encouragement and invaluable advise for both me, and the work, made this study possible. I am grateful to all of the co-authors for their valuable work. In particular, I would thank Professors Sami Franssila, Tapio Kotiaho, and Dr. Lauri Sainiemi. I want to especially express my appreciation to Lauri who has spent countless hours in the cleanroom fabricating the microchips that were used in these studies. Very special thanks are due to my former and present colleagues of the Division of Pharmaceutical Chemistry for making the work fun and even enjoyable; So in particular thanks are due to Anu, Inkku, Kati H., Laura L., Laura H., Linda, Markus, Mikko, Niina, Nina N., Nina S., Päivi U., Raisa, Sirkku, Tiina K., Tiina S. and Timo. Warm thanks to my parents Pirkko and Pentti for always nurturing me and teaching me the most important things in the world. Thanks also to my sisters and brothers for making my life happy, challenging and worth living when I was a child. I reserve my deepest and warmest thanks for my love and wife, Taija-Tuulikki, who always waited for me to return home from work, and gave me the best possible support at home. Without your encouragement and love, this work would not have been able to be finished. I also want to thank my children Vesa, Eero, Leo and Iida for their ability to make life eventful and giving me an excellent counterbalance to work. IV Contents ABSTRACT ........................................................................................................................III ACKNOWLEDGEMENTS .................................................................................................. IV CONTENTS ....................................................................................................................... V LIST OF ORIGINAL PUBLICATIONS................................................................................. VIII ABBREVIATIONS ............................................................................................................. IX 1 INTRODUCTION ............................................................................................................. 1 1.1 MINIATURIZED ELECTROSPRAY IONIZATION MICROCHIPS COMBINED WITH MASS SPECTROMETRY (MS) ..................................................................................................................................... 1 1.1.1 Electrospray ionization ..................................................................................... 1 1.1.2 Miniaturized electrospray ion sources ............................................................... 2 1.2 MINIATURIZED AND ON-CHIP INTEGRATED LIQUID CHROMATOGRAPHY ELECTROSPRAY IONIZATION MICROCHIPS ..................................................................................................................... 9 1.3 MICROREACTORS COMBINED WITH MASS SPECTROMETRY ..................................................... 12 1.4 MICROPILLAR ARRAYS IN ANALYTICAL APPLICATIONS ............................................................ 17 1.5 REFERENCES .............................................................................................................. 23 2 AIMS OF THE STUDY .................................................................................................... 30 3 SILICON MICROPILLAR ARRAY ELECTROSPRAY CHIP FOR DRUG AND BIOMOLECULE ANALYSIS (I) ................................................................................................................... 31 3.1 INTRODUCTION .......................................................................................................... 31 3.2 EXPERIMENTAL .......................................................................................................... 32 3.2.1 Chemicals and samples ................................................................................... 32 3.2.2 Fabrication ..................................................................................................... 33 3.2.3. Mass spectrometry and methods ................................................................... 34 3.3 RESULTS AND DISCUSSION ............................................................................................. 36 3.4 CONCLUSIONS ........................................................................................................... 40 4 FABRICATION AND FLUIDIC CHARACTERIZATION OF SILICON MICROPILLAR ARRAY ELECTROSPRAY IONIZATION CHIP (II) ............................................................................. 43 4.1 INTRODUCTION .........................................................................................................